The need for the ability to carefully track a wide variety of analytes in clinical medicine is ever increasing. Tracking may determine the metabolism of pharmaceuticals after administration, monitor the biological status quo, determine the presence of tumor cell markers, indicate the presence of tissue damage, etc. The quantitation of analytes in complex biological samples is complicated by the; interference of other constituents which may be sticky, have analogous structures, affect the detection of the label, and the like.
Methods which utilize high pressure liquid chromatography (HPLC) are useful for small organic molecules, but may not be able to resolve complex macromolecules such as glycoproteins. In order to quantitate low concentrations of such macromolecules in complex biological samples, it is normally necessary to use a reagent, often labeled, which specifically binds the analyte. After separation of bound from unbound reagent, the amount present can be quantitated by use of the label. Immunoassays such as ELISA, RIA or solid phase immunoassay have been used in clinical analyte analysis. They have the disadvantages of being time-consuming and lacking sensitivity in the very low range of analyte concentration.
Capillary electrophoresis is a highly efficient method for the separation and detection of molecules. Conventional methods of electrophoresis are limited by the heat induced during a run. Capillary tubes, in contrast, can be run at very high voltage gradients, due to their excellent heat transfer ability. The capillary tubes used are hollow silica glass with polyimide coating on the exterior to prevent breakage. The silica wall gives a net negative charge to the inner surface. The action of an electric field on positive counterions next to the negatively charged inner wall causes the bulk flow of liquid known as electro-osmotic flow (EOF). Separation of molecules is a combined result of the effects of EOF and preferential electrophoretic mobility.
Free solution capillary electrophoresis runs the sample into a single, continuous buffer. Electrolyte buffers may be simple salts, such as borate and phosphate, or may contain additives. Micellar electrokinetic capillary chromatography adds detergents above their critical micellar concentration, thereby allowing the separation of neutral molecules on the basis of hydrophobicity. Other available methods are adapted from slab gel electrophoresis, such as isoelectric focusing, which resolves the samples by isoelectric point, or the addition of linear or cross-linked polymer to allow molecular sieving to take place. When clathrates are added, stereoisomers may be separated.
A major advantage of capillary electrophoesis is the speed in which components may be capable of being resolved, coupled with reproducibility and high level of sensitivity. It is therefore desirable to provide methods for clinical sample analysis which can take advantage of these properties.
Relevant Literature
A review of capillary electrophoresis may be found in Landers, et al. (1993) BioTechniques 14:98-111. U.S. Pat. No. 5,120,413 describes the use of a borate buffer system in the capillary zone electrophoresis of glycoproteins in clinical samples. The resolution of hemoglobin variants by capillary zone electrophoresis is disclosed in U.S. Pat. No. 5,202,006.
Nielson, et al. (1991) J. Chromatography 539:177 describe a comparison of capillary zone electrophoresis, slab gel electrophoresis and HPLC for the separation of human growth hormone and antibody complexes. Schultz and Kennedy (1993) Pittsburgh conference March 7-12 report the use of fluorescence to detect insulin in capillary electrophoresis based competitive and noncompetitive immunoassays. Shimura and Karger (1994) Anal. Chem. 66:9-15 describe the resolution of immune complexes of recombinant human growth hormone and antibody by capillary isoelectric focusing, and detected by laser induced fluorescence.